Processes and apparatus for carrying out specific binding assays

ABSTRACT

A process for carrying out a specific binding assay (for example an immunoassay) in which (a) a sample under assay, possibly containing a substance being tested for, is reacted with (b) a specific binding partner for the substance being tested for, immobilised on a solid support, and (c) a specific binding partner for the substance being tested for which is conjugated to a detectable marker, thereby to form a complex by reaction between whatever quantities are present of the substance being tested for with reagents (b) and (c), in which the marker is immobilised to the support via the substance being tested for, and is detected or assayed as an index of the quantity present in the sample (a) of any of the substance being tested for; characterised in that reaction ingredients (a), (b) and (c) are all mixed in a single step for reaction in a single reaction liquid, and competitive interference between the binding reactions of the substance being tested for and reagents (b) and (c) is avoided either by use of an antibody of narrow and different, non-interfering specificity, such as a monoclonal antibody, to avoid the interference, or by use of a slow-release form of reagent (c).

This is a continuation of application Ser. No. 08/275,755, filed Jul.19, 1994, now U.S. Pat. No. 5,610,077, which is a continuation of Ser.No. 06/744,658, filed Jun. 14,1985, now abandoned, which is acontinuation of Ser. No. 06/348,048, filed Jan. 29, 1982, now abandoned,the last-mentioned application being a Section 371 filing of PCTapplication GB81/00107, filed Jun. 22, 1981.

This invention relates to processes for carrying out specific bindingassays, e.g. immunoassays, and to apparatus, e.g. test kits, forcarrying out these processes.

In particular embodiments the invention is applicable to enzyme-linkedand fluorescent-marker-linked specific binding assays, includingimmunoassays.

A wide variety of immunoassays and other specific binding assays isalready known.

Examples of such assays and the materials used for them are given in S.Spector, Ann.Rev.Pharm. (1973) 13, 359-70 (radioimmunoassays), L. E. M.Miles and C. N. Hales, Nature (1968) 219, 186-189 (assays usingradioactive, enzyme and other markers), E. Habermann,z.klin.Chem.u.klin.Biochem. (1970) 8, 51-55 (radioactively-labelled andenzyme-labelled assay procedures), and in G.B. patent specification No.1,363,565 (enzyme-labelled immunoassays) and U.S. Pat. Nos. 4,150,949and 4,160,818 (fluorescence-labelled immunoassay).

An important group of such assays comprises those in which a ternarycomplex is formed between a specific adsorbent, the material underassay, and a marker-conjugated material with specific binding capacityfor the material under assay (“conjugate”). Certain assays of this kindhave been called “sandwich” or “antiglobulin” assays. They have theproperty that the quantity of marker becoming fixed to the specificadsorbent is directly rather than inversely related to the quantity ofthe material under assay that participates in the ternary complex, andthis can be simply measured after separation of the immobilised materialfrom the remaining free marked conjugate, in whatever manner isappropriate to the marker in use.

However, as appears for example from the above-mentioned Habermann(1970) publication and G.B. specification No. 1,363,565, the performanceof these assays is not without difficulty: they demand either a numberof successive manipulation steps to carry out the assay reactions, orelse suffer from low sensitivity, which careful choice of reagents hasnot so far been able to overcome.

Commercial test kits are available comprising essentially:

(a) a plate consisting of an array of tubes or pre-formed wells whichare coated with an antibody or antigen as the case may be;

(b) an enzyme linked to an appropriate antibody (a so-called conjugate)against a substance to be detected if present in a test sample and

(c) a substrate for the determination of the activity of the enzyme.

One standard procedure for conducting an assay for antigen or antibodyinvolves:

(1) determining the working dilution for the test sample;

(2) removing any excess of antibody or antigen used to sensitise thewells;

(3) washing the wells;

(4) introducing a proportion of the suitably diluted test sample;

(5) incubating for about two hours to allow the substance to be detectedin the test sample to bind to the sensitising substance;

(6) washing the wells to remove unreacted material;

(7) introducing the suitably diluted conjugate (incubate for about 2hours);

(8) washing the wells to remove unreacted material;

(9) adding a solution of the substrate;

(10) incubating until a suitable intensity of colour develops as aresult of the reaction of the substrate with the enzyme;

(11) stopping the reaction, e.g. with a strong alkali and

(12) measuring the optical density of the reacted substrate solution.

This procedure is also time-consuming since each of the severalantibody-antigen reactions requires several hours to reach equilibrium.In practice, shorter incubation times are used but only at the expenseof sensitivity and/or economy.

According to the results of the present work, it is believed that anobstacle to the use of fewer assay steps is an unwanted interferencewith the formation of the desired immobilised complex which canoriginate in interfering reactions between two of the components. Byusing a specific binding agent of selected narrow specificity, or inslow-release form, such interference can be avoided, andhigh-sensitivity assays carried out using fewer manipulation steps.

According to this invention there is provided a process for carrying outa specific binding assay (for example an immunoassay) in which (a) asample under assay, possibly containing a substance being tested for, isreacted with (b) a specific binding partner for the substance beingtested for, immobilised on a solid support, and (c) a specific bindingpartner for the substance being tested for which is conjugated to adetectable marker, thereby to form a complex by reaction betweenwhatever quantities are present of the substance being tested for withreagents (b) and (c), in which the marker is immobilised to the supportvia the substance being tested for, and is detected or assayed as anindex of the quantity present in the sample (a) of any of the substancebeing tested for; characterised in that reaction ingredients (a), (b)and (c) are all mixed in a single step for reaction in a single reactionliquid, and competitive interference between the binding reactions ofthe substance being tested for and reagents (b) and (c) is avoidedeither by use of an antibody of narrow specificity, such as a monoclonalantibody, to avoid the interference, or by use of a slow-release form ofreagent (c).

The narrow specificity required of the antibody is a capacity to bindspecifically with the substance under test but without preventing thebinding reaction between the substance under test and its other specificbinding partner. Such an antibody can be selected out of a number ofantibodies with an affinity for the substance under test, by usingnormal methods to verify the progress of a binding reaction between theother specific binding partner and a complex previously formed betweenthe substance to be tested in the assay and the narrow-specificityantibody to be selected.

According to a preferred embodiment of the present invention, theconjugate between antibody and the enzyme or other marker, and/or theantibody (if any) which is coupled to the solid surface, comprises amonoclonal antibody or other antibody of sufficiently narrow specificityto ensure that the desired assay reaction or reactions are not impededby competition between the conjugate and the immunosorbent in theirreactions with whatever quantities are present of the substance beingtested for in the sample under assay. Monoclonal antibody ofsufficiently narrow specificity can, for example, be produced asantibody derived from a line of antibody-producing cells, derived from asingle antibody-producing progenitor cell or cells. Such a line can, forexample, be produced by known cell fusion, culture and isolationtechniques using very pure antigens as comparative material.

Alternatively, in many cases antibody of sufficiently narrow specificitycan be obtained in the (polyclonal) immunoglobulins of antisera raisedagainst discrete chemical or physical molecular fragments of thematerial under test, for example, antibody against Fc fragments (oragainst smaller peptide fragments) of immunoglobulins to be tested for,or against sub-units or peptides of protein antigens to be tested for.The object in each case is to ensure substantial freedom frominterference which can arise particularly, for example, in carrying outimmunoassays of the “sandwich” or “antiglobulin” test configurations.

In a “sandwich” test configuration, antigen under test can bespecifically adsorbed to a first antibody bound to a solid surface, anda second antibody carrying an enzymic or other (e.g. fluorescent orradioactive) marker is specifically bound to the adsorbed antigen undertest. Marker specifically so bound is used for measurement anddetermination of the antigen under test, e.g. by direct measurement,such as radiometry or fluorimetry, or exposure of enzyme marker tosubstrate followed by product measurement. Thus, in preferred sandwichtests, the two antibodies used can have different, non-interferingspecificity with respect to the same antigen under test.

In an “antiglobulin” test configuration, sometimes also referred to as a“sandwich” test configuration, the position is analogous: the materialunder test is itself an immunoglobulin; the material bound to a solidsurface is its corresponding antigen or hapten; and the materialcarrying the marker is an antiglobulin corresponding to the species andimmunoglobulin type of the antibody under test. In preferredantiglobulin tests, the antiglobulin can have sufficiently narrowspecificity as not to interfere with the subsequent adsorption of itscorresponding globulin to the insolubilised antigen.

If antibodies from ordinary antisera raised against unmodified antigen(polyclonal antibodies) are used in sandwich or antiglobulin tests,there is a very likely risk that if all ingredients are mixed in asingle step there will be interference between the two specificadsorption reactions. When such tests are carried out according to thepresent invention, using apparatus as described herein, suchinterference can be avoided either by using antibodies of narrowspecificity as described, or else by ensuring that the binding of testmaterial to the solid surface takes place before exposure of testmaterial to the other (marker-conjugated) binding agent if there is arisk that binding by that other agent would prevent subsequentadsorption to the solid surface. Such a sequence can be ensured byarranging for slow release of the other (marker-conjugated) bindingagent.

Particular instances of suitable assay specificities, antibodyspecificities, and slow-release forms of conjugated reagent (c) aredescribed for example below.

It has also been found that in carrying out such specific bindingassays, a worthwhile improvement in reaction kinetics can be obtained ifthe reaction liquid containing ingredients (a), (b) and (c) is containedin a well or cup of which the majority of the volume is occupied by adisplacer body. (The use of inserts of various rod or ball shaped formsis known in connection with other kinds of immunoassay, as described inG.B. Specification Nos. 1,414,479 and 1,485,729.)

The displacer body can, for example, be of a shape substantiallycomplementary to and slightly smaller than that of the cup or well, sothat the liquid phase containing one of the specific binding reagents isapproximately in the form of a shell occupying the space between thedisplacer and the cup or well. The displacer can be loose-fitting andnot fixedly mounted, i.e. movable relatively to the cup or well, so thatby relative motion between displacer and well the liquid between themcan be given a stirring or agitation motion.

For example, a round well can have a round displacer therein with anexternal diameter slightly smaller than the diameter of the well. Thepresence of the displacer can reduce the space available for liquid inthe well by a factor of for example 2-10, e.g. 3-8, comparing volumesbased on similar liquid levels in the well, e.g. when filled to itsnormal operating level, or its maximum capacity. For example, amicrotitre well designed to have 300 microlitre of liquid filled into itduring a normal assay, can be used with a displacer leaving 30-150microlitre liquid space, e.g. 50-100microlitre.

The use of wells or cups together with displacers as described hereincan improve the efficiency of the assay reaction steps because, in thefirst place, it allows more concentrated reagents to be used with noincrease in the weight of reagent or decrease in the size of themicrotitre wells, compared with the normal conditions encountered inmicrotitre wells of given size; and in the second place, it increasesthe sensitised surface area available to react with a given liquidreagent volume, so that comparatively faster adsorption kinetics can beachieved without having to increase specific reagent density on thesensitised surface or encountering problems of crowding.

A set of displacer bodies can be preferably present in certainembodiments of the invention, e.g. as an integral part of a lid whichcan be fitted onto a microtitre plate, e.g. a standard plate of 8×12wells. The set can be large enough to fit all the wells of the plate ora sub-set thereof, e.g. a row. The dimensions of the displacers and thevolume of liquid to be dispensed into the well can be chosen relative tothe well in the manner described above, and preferably so that theliquid to be tested is in contact with substantially the major part andpreferably the whole inner surface of the well.

The immobilised specific binding partner (reagent (b)) for the substanceto be assayed can be immobilised on the wall of the well or cup in whichthe assay reaction takes place. Alternatively, according to a feature ofthe invention independently capable of providing advantage andconvenience in use, a liquid displacer, for example in the form of astick, peg or stud, for dipping into a liquid assay reagent, can have animmunosorbent surface. This allows the portion of the assay materialsneeding to be carried over from one reagent to the next, and theassociated manipulations, to be handled more easily than when thesensitised surface is part of a hollow well. An alternative form forsuch a liquid displacer body is a tuft of bristles or leaves of suitablematerial, or equivalent body with large surface area. A furtheralternative form is a stud or peg with relatively hollowed-out andprojecting portions of its surface, e.g. with grooves and associatedribs, e.g. annular grooves. Such an arrangement can give robustness,increased sensitised surface area, and better reactivity.

Test apparatus according to related embodiments of the invention canthus comprise a set of sensitised liquid-displacer bodies of one or moreof such forms, joined to a common handling-bar, link or lid, and for usein combination with a complementary set or sets of wells containing anyof the remaining materials used in the assay. The several displacerbodies of the set can have the same or different sensitisation so thatone or a plurality of different assay types can be carried throughsimultaneously. If desired, the displacer bodies can be removably andexchangeably mounted on the handling bar, link, or lid, so that sets ofdesired specificity can be built up at will from a common stock forcarrying out large numbers of tests according to a desired pattern.

One advantage of such arrangements is that a number of displacer bodiescan be sensitised in the same body of liquid ragent, avoidingfluctuating conditions of concentration, etc., resulting from dosingaliquots into wells.

The displacer bodies can, in this embodiment, be of any materialsuitable for the preparation of an immunosorbent by covalent bonding oradsorption: e.g. polystyrene, nylon, or cellulose acetate. (The natureof the displacer surface does not matter provided it is inert, when thesensitisation is to be on the well surface rather than the displacersurface.) Linkage of antibodies, antigens, etc., to the displacer bodiescan be carried out by linking methods known in themselves, e.g. partialacid hydrolysis of nylon surface, substitution of exposed amine surfacewith glutaraldehyde, and coupling of material to be bound, e.g. antibodyor antigen, to immobilised aldehyde groups. Suitable methods among awide variety are given for example by Inman & Hornby (1972) Biochem.J.129, 255; Campbell, Hornby & Morris (1975) Biochim,Biophys.Acta 384,307; Mattiasson & Nilsson (1977) F.E.B.S. Letters 78, 251; and G.B.patent specifications Nos. 1,470,955 and 1,485,122.

It can be seen that the invention also provides a kit of test materialsfor carrying out a specific protein-binding assay, comprising (i) animmobilised specific binding partner for a substance to be tested for,carried on a solid support, and (ii) a marker-conjugated specificbinding partner for the substance to be tested for, which can be addedto a reaction liquid contacting immobilised reagent (i) either as aslow-release form, or in any form provided that the specific bindingpartners in reagents (i) and (ii) include an antibody of narrowspecificity so that reagents (i) and (ii) do not interfere with eachother's binding reactions with the substance to be tested. Optionallythe kit can also comprise materials for later estimation of the amountof marker immobilised during the assay.

Reagent (i) can be immobilised on either a displacer body for a reactionwell, or on a reaction well wall, as described above. A slow-releaseform of reagent (ii) can be for example a sucrose or equivalent glaze ona complementary surface of either the displacer or the well wall, alsoas described above. The narrow-specificity antibody can be selected forexample from monoclonal antibodies in the manner already described.

The invention is further illustrated by reference to the followingdetails and accompanying drawings, of which:

FIG. 1 is a vertical cross-section of an arrangement of container anddisplacer for carrying out specific binding assays according to theinvention.

FIG. 2 diagrammatically shows an array of several of the arrangementsshown in FIG. 1.

FIGS. 3 and 4 show in diagrammatic side view and part-sectional endview, an assembly of displacers for carrying out certain embodiments ofthe invention.

FIG. 1 of the drawings shows in vertical cross-section a round-bottomplastics microtitre well-shape vessel 1 containing a round-sectionround-bottom displacer body 2 of slightly smaller and complementaryshape. The displacer 2 can be of glass or plastics material. The normalcapacity of well 1 is for a 300 microlitre sample. The presence of thedisplacer 2 reduces the effective capacity to 50 microlitre. Thisquantity of liquid 3 forms a shell in contact with the whole of thesurface area of the normal capacity of the well and with the displacerbody 2. Thus the surface-to-volume ratio of liquid 3 is several timesgreater than that of the body of liquid which would fill the normalcapacity of the well in the absence of displacer 2.

Displacer 2 can be left loose so that during use of the well for assayany agitation given to the well 1 causes stirring of liquid 3.Alternatively, displacer 2 can be directly driven. In one arrangements,shown in FIG. 2, a tray 4 containing a 2-dimensional array of wellssimilar to well 1 has a lid 5 to which a complementary set of displacerbodies similar to cisplacer 2 is mounted the displacers may beintegrally formed with lid 5, e.g. in the form of hollow mouldedprotrusions. In an alternative arrangement the fixed set of displacersmay form a row or rows only, less than the full array.

In use, for example, for immunoassay of the ELISA type, the bottoms andlower parts of the walls of wells 1 are coated with immunologicalreagent to form immunosorbent, and 50 microlitre quantities of thematerials under test, in suitable dilutions and media, are contactedwith the well walls and bottoms, by the use of the displacer bodies 2.Immunological reagent conjugated to enzyme marker, if not alreadydispersed throughout the reaction liquid, is added to the reactionliquid in an alternative way: The displacers can be coated with aconjugate of a detectable enzyme or other marker with an immunologicalreagent, e.g. antigen or antibody. Suitable quantities of the conjugatecan be dried onto the tips of the displacer bodies, preferably in aglaze, e.g. of sucrose, to prevent aggregation of conjugate moleculesand ensure a gradual and slow solubilisation of the conjugate therebyavoiding saturation of the immunosorbent with the conjugate before theformer gets the chance to react with the substance to be detected. Thetest is then performed by dispensing an appropriate volume of testsample consisting, for example, og patient's serum into a well, followedby inserting a displacer body into the well. Thus, the sample andconjugate can be added almost simultaneously, thereby reducing thetime-consuming sequence of operations. After the slow release of theconjugated material from the dried glaze on the tips of the displacers2, formed for example, by evaporation of a solution of the conjugate andsucrose, (or after formation of the assay complex from predispersedreagents) the amount of specifically bound enzyme marker is measured inthe usual way. The dosimetry and quantitation of the reagents for thetest are carried out according to well-established methods and inthemselves form no part of the invention.

FIGS. 3 and 4 of the accompanying drawings show, in diagrammatic sideview and end (part-sectional) view, an assembly of displacer bodies forcarrying out specific binding assays according to a further andadvantageous arrangement given by way of example. The assembly of suchdisplacer bodies can form part of composite apparatus according to theinvention comprising such an assembly (or a single such displaceraccording to the invention) together with a complementary well or wellsfor carrying the liquids used in the assays. Either (preferably) thedisplacer, or the well, carries immobilised specific binding agent.

Referring to FIGS. 3 and 4, the characteristic component of theapparatus for carrying out the assay in each well is a liquid displacer11 in the form of a stud, stick or peg as shown for dipping into liquidin an assay well as described herein. The displacer 11 is formed, e.g.by moulding or turning in a suitable material such as nylon, celluloseacetate or polystyrene. One preferred form is a nylon peg, of forexample, about 5 mm greatest diameter, or slightly narrower than theinternal diameter of an associated microtitre well, and about 15-20 mmheight to project out of such a well. About, for example, 8 millimetreof a lower extremity 12, or other lower portion of displacer 11, canconveniently be provided with grooves, a screw thread, chamfering, orother irregularity to increase its surface area, not shown in detail inthe drawings, which in FIGS. 3 and 4 indicate only a slight overallnarrowing of grooved extremity 12 of displacer 11 due to the formationof such grooves.

Each displacer 11 is mounted on a handling piece 13 which can haveeither one displacer 11 or as many as desired mounted thereon, e.g. in aone-dimensional array, as shown, or a two-dimensional array,corresponding to the arrangement of FIG. 2. The displacers 11 can beformed either as one piece with the handling piece or as separatemountable parts which can, if desired, be exchangeable. Illustrated is asocket-and-screw arrangement for exchangeably mounting displacers 11 ofthe type described at, for example, about 9 millimetre intervals,corresponding to well spacings in the complementary microtitre plate.The handling piece can be of any suitable material, e.g. plastic such asnylon, etc., or metal, e.g. brass, copper or stainless steel.

Biological/chemical conditioning of the displacer 11, e.g. the lowergrooved extremity 12 thereof, can be carried out for example as followsto suit the material for carrying out specific binding assays such asimmunoassay. The displacer can, if desired, be used as described inExample 1, but alternatively and preferably, it can be made to carry animmunosorbent or other specific binding adsorbent. Antibody or antigen,for example, of type suitable for the desired assay configuration whichin itself forms no part of this invention, can be attached by physicaladsorption (especially when using polystyrene) or (preferably) bycovalent linkage to grooved extremity 12, e.g. by methods alreadyreferred to herein.

The assay is then carried out, for example, as follows. Reagent and/ortest solution is placed in the well, to a volume sufficient to fill thewell only when the displacer is inserted (50-200 microlitre forexample). With the displacer present the binding reactions take place,and washing can be achieved simply by removing the displacers andrinsing them, e.g. under running tap water.

Even though two binding events may be required (e.g. test antibody bindsto immobilised antigen, then enzyme-conjugated antiglobulin binds to thetest antibody) it is possible to complete the binding sequence in asingle step. This requires the careful choice of reagents, as describedabove, so that the conjugate antibodies do not compete for all thebinding sites on the test material and prevent it from binding to theimmunosorbent.

The presence of the displacers in the wells can provide for theimportant operation of stirring the fluid samples. Not only does thisallow a more rapid binding reaction, it also allows the conjugate to bepre-dried into the wells in a firm and stable state, such that it canonly be reconstituted by means of such stirring. The stirring might becontinued for the duration of the whole binding step, or might bestarted only after the first binding reaction has been completed. Asuitable form of coating is for the well bottoms to be given a driedsucrose glaze containing the conjugate. This can release slowly enoughto achieve the desired sequence of binding reactions, as discussedherein above.

After the binding reactions have been completed the displacers can bewashed and transferred to another set of wells containing a solution ofenzyme substrate, where they remain until the positive controls havedeveloped an adequate colour (and negative controls are still adequatelyblank). In a standard ELISA, the enzyme/substrate reaction has to bestopped by adding a reagent such as 2M sodium hydroxide solution, buteven this step can be dispensed with when the arrangement of thisinvention is used. The reaction can be terminated simply by removing thedisplacers from the substrate solutions; the substrate-containing wellscan then be simply placed in a standard micro-ELISA reader for themeasurement and recording of results. In existing commercial ELISAsystems the immunosorbent component (usually the microtitre wells orequivalent) is necessarily a disposable item, but in a system accordingto this invention, possible covalent attachment or antigens can allowthe immunosorbent to be re-usable (if it is desired to be so). Thebinding reactions can be reversed by soaking the displacers in areleasing agent such as 4M magnesium chloride, or sodium dodecylsulphate, whilst the covalently attached reagent remains intact. Thisarrangement can bring benefits in terms of precision, reliability, speedand/or sensitivity. For the manufacturer and user alike there can beimportant economies in the use of immunoreagents which are often scarceand expensive.

A further optional feature which can be present in the above-describedformat of this invention is that of the precision heating of individualreaction mixtures. The rates of the binding and of the enzyme/substratereactions are dependent on temperature; temperature differences across amicrotitre plate can cause related response variations, so creatingserious defects in calibration and reproducibility. Normally, ELISAreactions are speeded up by placing the tray in a standard airincubator, but this can be less than satisfactory, because the rate atwhich wells are heated often depends on their position in the tray. Therate of heat exchange in an air incubator is rather slow: the wholeplate warms up relatively slowly. Individual heaters can be made (forexample, from precision resistance thermometers used in a heating moderather than a measurement mode), and inserted into the centre of thedisplacer bodies, for example those illustrated in FIGS. 1-4, and, inthat position, can ensure a rapid and efficient delivery of heat throughthe displacer body surface. This system (which incorporated suitablethermostatic control of a kind well known in itself) can thereforeprovide faster reactions and reduce well-to-well variation at the sametime as dispensing with the need for an incubator.

EXAMPLE 1

An illustrative scheme of preparing materials for immunoassay andcarrying out the assay according to the invention is described below,for use with the displacers shown in FIGS. 3-4. The particular assayconfiguration illustrated happens to be an enzyme-linked antiglobulintest directed to assaying antibodies against soya protein. It will beclear that other configurations and specificities can be used and thearrangements of this invention applied to them, as for example many ofthose mentioned in U.S. Pat. Nos. 3,654,090, 3,971,932, 3,839,153,3,850,752, 3,879,262 and 3,996,345.

Preparation of Soya Protein-linked Displacer Pegs

Nylon pegs as described above in relation to FIGS. 3-4 are immersed in3.6M HCl and held at 48° C. for 35 minutes after which they are washedunder running tap water and then soaked in distilled water. Next, theyare immersed in chilled 12.5% aqueous glutaraldehyde and kept at 4-8° C.for 20 minutes. This reaction is stopped by washing the pegs underrunning tap water and then soaking them in a large volume of distilledwater for at least 2 hours.

After this activation sequence, the pegs are placed in the wells of amicrotitre tray, together with 200 _(/)ul of a solution of soya protein(300 _(/)ug per ml, in phosphate buffered saline with Tween detergent(PBST)+0.1% thimerosal). The trays and pegs are placed in a sealedcontainer and the reaction continued at 37° C. for 54 hours.

Finally, unused active sites are blocked by transferring the pegs intoanother set of wells containing 200 _(/)ul of 1.5M ethanolamine, wherethey are maintained at room temperature for 24 hours. Before use, theyare soaked in PBS+0.15% Tween for a further 3 days at room temperature.

In an alternative preparative method, the hydrolysis and glutaraldehydetreatment are identical but, instead of 300 _(/)ug/ml of protein beinglinked to the surface, the pegs are treated with poly-L-lysine (PLL) at100 _(/)ug/ml for 18 hours at 37° C. After a thorough wash in tap waterand then PBST, the linked PLL is activated by treatment with 12.5%glutaraldehyde (18 hours at 37° C.). Finally the soya protein is linkedto the glutaraldehyde-substituted PLL by incubating the treated pegs in200 _(/)ul of soya protein at 100 _(/)ug/ml, for 18 hours at 37° C.Excess active groups are blocked by treatment in 1.5M ethanolamine at pH8.5 for seven hours.

In further alternative procedures, the nylon can be treated withdimethyl-1,3-propane diamine to cleave nylon polymer chains, the freeamino groups being then substituted with glutaraldehyde, and optionallyafter further insertion of amino and aldehyde “spacer” groups, theprotein desired to be coupled is linked to the terminal aldehyde.

In a further alternative method, not involving polymer chain cleavage ofthe nylon, displacers are soaked in PBS+2% sodium dodecylsulphate andthen allowed to dry in the air at room temperature overnight. They areO-alkylated by being immersed in 100% dimethyl sulphate for 3 minutes atroom temperature. The reaction is stopped by washing the pegs in icecold ethanol and then cold water. Immediately thereafter the pegs areimmersed in a solution of soya protein (100 _(/)ug/ml) and kept at roomtemperature for 3 days. Finally they are transferred to a 10 mM solutionof ethanolamine where they are maintained at room temperature for afurther 2 days, before a final wash in Tris/HCl buffer, pH 8.0.Alternatively, the alkylating agent used can be triethyloxoniumtetrafluoroborate, as described by Inman and Hornby (1972) cited above.Suitable coupling methods from which choice can be made are mentionedin, for example, GB Specifications 1,316,990, 1,470,955, 1,485,122-3,and U.S. Pat. No. 3,817,837 (cols. 31-34, for example).

Preparation of Enzyme Labelled Sheep-anti Rabbit IgG

Rabbit globulin (prepared from rabbit serum by sodium sulphateprecipitation) is immobilised onto cyanogen bromide-activated sepharose4B (Pharmacia) by the normal procedure described by the manufacturers. Ahigh-titre sheep antiserum to rabbit globulin (Seward Laboratory,Immunostics (Trade Mark) Product BS 01) is passed down the column andthe non-bound fall through fraction rejected. The bound antibodies arereleased by passing 0.5M acetic acid down the column. Theprotein-containing fractions are pooled and dialysed against PBS to givean antibody preparation which is conjugated with enzyme by the followingprocedure.

The following method is based on that described by Engvall and Perlmann(1971) (Immunochemistry, 8, 871). The IgG fraction of sheep antiserum torabbit IgG (Seward Laboratory, Immunostics (Trade Mark), Product BS 01)is adjusted to a concentration of 5 mg/ml. A 0.1 ml aliquot of this isused to dissolve 1.5 mg of Sigma type VII alkaline phosphatase (suppliedas a suspension in 3.2M ammonium sulphate). The resultant solution ifdialysed against phosphate buffered saline (PBS) and then mixed with 5_(/)ul of a 25% glutaraldehyde solution (aqueous). The reaction withglutaraldehyde is continued for 2½ hours at room temperature and thenstopped by dilution to 1 ml with PBS and extensive dialysis. Finally,the produce is diluted to 10 ml in 0.05M tris/HCl buffer (pH 8.0)containing 50 mg/ml ovalbumin, 0.2 mg/ml magnesium chloride, 0.2% sodiumazide and 0.2% merthiolate. The conjugate can be applied to a microtitrewell bottom by making it concentrated in sucrose solution and a 5-50microlitre aliquot is then evaporated to a hard sucrose glaze at 37° C.for slow release on stirring.

Preparation of Soya Antigen

The protein used as an antigen is prepared from soya bean flour by amethod based on the procedure described by Koh (1978) - Can.Inst.FoodSci.Technol.J., 11, 124. Soya bean flour (10 g) is dispersed in PBS (100ml) and after thorough mixing the suspension is centrifuged to yield aclear supernatant solution.

An aliquot of this solution is mixed with 30 g of urea to give a finalvolume of 50 ml. This solution is heated in a steam bath for 1 hourbefore 1 ml of 2-mercapto-ethanol was added. Steaming is continued for afurther 45 minutes. After being cooled to room temperature the solutionis dialysed, first against running tap-water for 2 hours and thenagainst 2×5 litres 0.15M sodium chloride. The final dialysed solutionswells to 80 ml and has a very slightly opalescent appearance.

Preparation of Rabbit Antibodies to Renatured Soya Protein

Rabbits are given an intra-muscular injection of renatured soya antigen(2 mg) in Freunds' complete adjuvant emulsified as a multiplewater-in-oil in water system. The volume given to each animal is 2.0 ml.Subcutaneous booster injections of antigen in saline (0.25-0.5 ml) aregiven 31, 100, 108 and 115 days after the initial inoculation. Serumsamples are taken at various times through this schedule, and used assources of antibody to be tested for by the following assay schedule.

Immunoassay using the Sensitised (Soya-protein-linked) Displacers

Rabbit antisera and the sheep-anti rabbit/enzyme conjugate are dilutedappropriately in PBST and then the following sequence of operations wasfollowed.

1. Diluted antisera placed in wells, followed immediately by sensitiseddisplacer pegs.

2. Displacer pegs + wells (containing a sucrose glaze of conjugate) +sample placed in sealed container and incubated at 37° C. for about 90minutes. Agitation is carried out but only after about half theincubation period has elapsed.

3. Displacer pegs removed and rinsed under running tap water, then PBST.

4. Washed displacer pegs placed in wells containing substrate for enzyme(Sigma 104 (Trade Mark)).

5. Displacer pegs + wells + substrate placed in a sealed container andincubated at 37° C. for 45 minutes.

6. Displacer pegs removed and optical densities of substrate samplesmeasured.

The standardisation and calibration of this test is carried out bymethods which are in themselves well established and form no part ofthis invention.

EXAMPLE 2

An alternative assay and set of assay materials for the detection orestimation of anti-(soya) antibodies uses a monoclonal antibody. Thesematerials and the assay are carried out as Example 1 except that: Amouse monoclonal anti-(rabbit IgG) with good affinity is used in placeof the (polyclonal) sheep antiserum to rabbit IgG previously used.Instead of the IgG Traction of the antiserum prefiously to be mixed withthe enzyme and conjugated, an ascitic fluid fraction containing themonoclonal antibody is used: this fraction is prepared by affinitychromatography on the same kind of rabbit globulin-conjugated Sepharosegel as in Example 1, but the elution is carried out afterwards with 4MMgCl₂ solution instead of 0.5 M acetic acid. The final conjugatepreparation is added to the well directly in step 1 instead of beingapplied as a sucrose glaze, and the incubation time in step 2 can bereduced to 1 hour. It is found that this assay provides not only a gainin time and simplicity but can also yield a more accurate and reliablerelationship between final colour development in steps 5-6 and quantityof anti-(soya) antisera added in step 1.

EXAMPLE 3

An assay specific for K-type immunoglobulin G and the preparation ofmaterials for it can be carried out as follows.

Pegs moulded of nylon 66 in the form illustrated in FIG. 3 are washed inalcohol and distilled water. Mouse monoclonal anti-(human K-chain)antibody is immobilised on to them by exposure of the pegs at roomtemperature for a few hours of overnight to a 20 microgram/ml solutionof the antibody in 0.01 M aqueous phosphate buffer at pH 8, kept free ofother protein and of any detergent. The preparation of monoclonalantibody to K chain (and also of the antibody used below directed toγ-chain) is derived from a corresponding ascitic fluid by affinitychromatography on a column for example of Affigel (Biorad, Trade Mark)conjugated to human IgG, in otherwise the same way as in Example 1 or 2.(About 10 mg IgG is used for conjugation of 1 ml of gel, and at least 2ml of gel is used for chromatography of 1 ml of ascitic fluid.)

A mouse monoclonal (human γ-chain) antibody is conjugated with alkalinephosphatase as in Example 1, to form the other specific binding reagentin the assay.

The general procedure of Example 1 or 2, was then used together withthese materials, except that the non-covalent nature of the absorptionof antibody to form immunosorbent made it advisable to use as reactionbuffers at pH 7.1 phosphate/saline/Tween/ovalbumin buffer (0.15%phosphate, 0.85% NaCl, 1.5 ml/ml Tween, 1 mg/ml ovalbumin). Thisprovided a specific and sensitive assay for K-type human IgG. Analternative arrangement for the assay of this Example involves omittingthe conjugate from step 2, incubating the pegs and test sample about 60minutes, rinsing, then incubating the pegs about 60 minutes withconjugate dissolved in a similar buffer before going to step 3. Bothassay methods give advantage in allowing highly specific determinationof the γ-K subpopulation of IgG molecules, because the narrowspecificity avoids the interfering cross-reactions which would haverendered such specific assays impossible using less specific reagents.

It was found in one series of trials that the assay result could be madelinear in the range 10-100ng K-IgG per 0.2 ml assay reaction volume,(using about 50 ng conjugate based on starting antibody content).

A corresponding assay for γ-type IgG can be easily carried out if in thepreparation of the immunosorbent, i.e. the nylon 66 pegs with antibodyimmobilised thereon, a mouse monoclonal antibody direction against-chains is used instead.

It can be seen that the particular methods and materials described abovecan be varied in specificity to provide a wide range of assays sharingthe convenience of fewer manipulation steps and freedom from interferingside reactions in use.

Among the monoclonal antibodies that can be obtained according topublished procedures and selected for use as assay materials in thepresent invention are for example anti-rotavirus, anti-(human) IgE,anti-(bovine) IgG, anti-α-retoprotein, anti-(human and other) IgA,anti-(thyroid-binding globulin), anti-β₂ microglobulin,anti-pregnanediol, anti-ocstronediol and anti-(human) IgM antibodies,and others. According to the invention these reagents can be used toprovide the basis of highly-specific assays for the correspondingspecific binding partner which may correspond to the whole population ora specific subpopulation of the type of material under assay: any of theabove-described assay procedures are usable for this purpose.

Not all of the corresponding assays need be immunoassays in thestrictest sense. For example, using monoclonal anti-(TBG), acorresponding specific binding assay involves immobilised T₃ to T₄ asthe specific binding partner of the TBG being tested for, and the use ofa selected marker-conjugated monoclonal anti-TBG enables a sensitive“sandwich” type assay to be carried out in few steps withoutinterference between immune complex formation and the binding of TBG toT₃ or T₄.

An assay for specific immune complexes can be carried out in ananalogous way by the use of immobilised conglutinin and amarker-conjugated monoclonal antibody specific to the component of theimmune complexes desired to be assayed.

It will be well understood that the other arrangements of this inventionare also susceptible of wide variation and application. For example, aset of displacers as shown in FIGS. 3-4 can comprise a handling piece orholder fitted with, e.g., eight pegs, the displacers being appropriatelysensitised for use in the assay of human antibodies to any one or moreof, for example, toxoplasma, rubella virus, cytomegalovirus andherpesvirus. The displacers can have corresponding antigen fixedthereto, and a marker-conjugated anti-(human globulin) is used in thetest which is chemically of the known antiglobulin format, with reagentsprovided and calibrated accordingly. It can be specially convenient ifall four antigen sensitisations are represented in duplicate, each ontwo displacers of the assembly, shown in FIGS. 3-4. Then such anassembly can, for example, be carried through assay steps designed toassay eight aliquots of a single patient sample, in duplicate for eachof the four above-mentioned antibodies. Such an arrangement can beespecially convenient in that the remainder of the test tools andreagents, other than the displacers carrying specific sensitisation, canbe qualitatively similar for all the assays, for example, the conjugatecan be an anti-(human globulin) antibody coupled to a suitable markerenzyme, and common means can be used for detecting the enzyme. Thesecomponents can be suitably provided in microtitre wells prepared inconjunction with the sensitised displacer pegs, if desired.

Other immunoassay arrangements according to the invention can bedirected in specificity towards, for example, immunoglobulin E,(otherwise described as reagin-immunoglobulin), for which anantiglobulin assay can in format resemble the radioimmunoassay usingantiglobulin described in U.S. Pat. No. 3,720,760, with optionally thesubstitution of another kind of marker conjugating the anti-IgE or otherlabelled antibodies used, e.g. a fluorescent or especially an enzymicmarker, used as described above. Where markers other than enzyme markersare used, the appropriate respective known coupling techniques are used.Analogous assay arrangements can be made for clinical diagnostic use,e.g. plasma C-reactive protein, urinary β₂-microglobulin or Factor VIII.For these antigenic test materials it can be convenient to use asandwich-type assay format.

Other modifications and variations will be apparent to the skilledreader. In particular, the vesicle-bound marker systems described inspecification EP-0014530 can be used to provide marking for the assaysof the present invention. Reference is also made to this specificationand the literature cited there for further examples of sandwich andantiglobulin type assay configurations.

What is claimed is:
 1. In the process for carrying out a specificbinding assay comprising the steps of reacting (a) a sample under assay,possibly containing a substance being tested for, with (b) a specificbinding partner for the substance being tested for, immobilised on asolid support, and (c) a specific binding partner for the substancebeing tested for which is conjugated to a detectable marker, thereby toform a sandwich complex by reaction between whatever quantities arepresent of the substance being tested for with reagents (b) and (c) andimmobilising the marker to the support via the substance being testedfor, the marker being detected or assayed as an index of the quantity ofthe substance being tested for present in the sample (a), theimprovement which comprises using reagents (b) and (c) together forreaction with sample (a) and avoiding competitive interference betweenthe binding reactions of the substance being tested for and reagents (b)and (c) by using, as reagents (b) and (c), monoclonal antibodies each ofnarrow and different, non-interfering specificity.
 2. A processaccording to claim 1 wherein the reagent (b) is an immobilisedmonoclonal antibody or an immobilized substance to which animmunoglobulin under assay can specifically bind, and reagent (c)comprises a different non-interfering monoclonal antibody specific tothe binding partner of reagent (b).